The extracellular part of the influenza M2-protein (M2e), fused to Hepatitis B virus core protein, is an effective prototype vaccine against influenza A. It is universal, because the target, M2e, has been conserved in all human influenza A strains since 1933, despite epidemics and pandemics. The aim now is to enhance the efficacy and immunogenicity of the vaccine: (a) The present vaccine contains monomeric M2e-peptide. But the natural M2-protein is a tetramer. Therefore a vaccine will be developed which will present native-like, tetrameric M2e to the immune system. This is achieved by fusion of M2e-peptide to a tetramerising leucine-zipper (M2e-lz4), further linked to a carrier. The questions to answer are whether the tetrameric M2e-vaccine induces more effective protective antibodies, are these different from those reacting with the monomeric M2e, do they react better with (native) M2-protein on virus-infected cells, etc. (b) An influenza vaccine which can be administered intranasally has several advantages: (i) It may induce enhanced immunity in the respiratory tract region, and (ii) needle-free vaccination is expected to increase acceptability. To enhance the effectiveness of nasal M2e-vaccine, it will be linked to a ligand which will bind it to receptors on cells of the nasal cavity. E. coli heat-labile enterotoxin (LT) has the composition AB5, and the B5-domain binds strongly to GM1-ganglioside present on mammalian cells, including epithelial and immune cells of the nasal tract. M2e will be fused either at the N- or the C-terminus of LT-B or to LT-A2, the non-enzymatic, non-toxic C-terminal part of the A-chain. By further insertion of an Iz4-domain it should be possible to obtain a vaccine containing a tetrameric M2e-domain, physically linked to a strong cell-ligand. (c) An intranasally administered vaccine needs an appropriate adjuvant. Mutant LT (e.g. R192G) is effective as a mucosal adjuvant, but there is a concern for potential residual toxicity. Nils Lycke has described a very effective, non-toxic, new adjuvant, CTA1-DD, which unlike LT, only binds to B-cells. CTA1-DD will be evaluated in combination with all M2e-based structures described above. (d) An immunogen covalently linked to an adjuvant is expected to be more effective. The Oprl lipoprotein from Pseudomonas aeruginosa is a potent fusion partner for induction of Type I immunity. Both monomeric M2e and tetrameric M2e-lz4 will be fused to Oprl and tested for efficacy. [unreadable] [unreadable]